We propose to analyze the functions of the three proteins composing the nucleocapsid of vesicular stomatitis virus. Special emphasis will be placed on the role of these proteins in transcription and replication of the viral genome. We shall continue our analysis of in vitro transcription to determine why the RNA from the defective interfering T particle is not transcribed. The T particle and temperature sensitive mutants will be utilized to examine regulation of transcription at the molecular level. In vitro analyses will involve determining the stoichiometry of transcription, the positions and possible specialization of the initial polymerase binding sites, and the molecular interactions responsible for assembling the transcriptase within the virus particle. Hybridization and acrylamide gel electrophoresis of in vitro transcription products are planned to determine if purification of the transcriptase alters its specificity. Virus infected cells will be analyzed to define the template for viral RNA replication. Newly replicated viral RNA will be identified by molecular hybridization with the complementary products of the in vitro transcriptase assay. Finally, the nucleocapsid will be dissected into its component proteins and reassembled to assess the minimal number of proteins required to form an infectious particle capable of reproducing itself. Infectivity will be assayed by formation of plaques in the presence of DEAE-dextran.